Regulation of Staphylococcus aureus colonization and disease

金黄色葡萄球菌定植和疾病的调节

• 批准号: 10228660
• 负责人: ALEXANDER R HORSWILL
• 金额: $ 37.27万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228660
• 关键词: Address; Adherence; Adult; Antibiotic Resistance; Bacterial Adhesins; Binding; Binding Sites; Biochemical; Biological Assay; Cells; Chemotaxis; Cues; DNA Binding; Disease; Environment; Enzyme Kinetics; Exhibits; Family; Goals; Healthcare Systems; Human; Immune Evasion; In Vitro; Infection; Infectious Skin Diseases; Kinetics; Laboratories; Leucocidin; Life Style; Ligands; Membrane; Membrane Proteins; Modeling; Molecular Analysis; Mus; N-terminal; Nasal cavity; Output; Patients; Phenotype; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Population; Property; Quantitative Reverse Transcriptase PCR; Regulation; Regulon; Reporter; Risk Factors; Role; Signal Transduction; Site; Skin; Skin colonization; Staphylococcus aureus; Staphylococcus aureus infection; Surface; System; Testing; Time; Urease; Virulence; acute infection; antibiotic resistant infections; base; chronic infection; enzyme activity; extracellular; improved; in vitro Model; in vivo Model; in vivo imaging; inhibitor/antagonist; inorganic phosphate; insight; intravital imaging; methicillin resistant Staphylococcus aureus; mutant; neutrophil; opportunistic pathogen; pH Homeostasis; pathogen; prevent; promoter; response; sensor; small molecule libraries; trait; transcriptome sequencing; vaginal mucosa

Project Summary Staphylococcus aureus is an opportunistic pathogen that causes a broad spectrum of acute and chronic infections. Antibiotic resistance levels are growing and methicillin-resistant S. aureus (MRSA) infections are more challenging to treat, resulting in increased burden on both patients and healthcare systems. Despite being such an effective pathogen, S. aureus can asymptomatically colonize approximately 20% of the healthy adult population, primarily in the nasal cavity and secondarily on the skin. There is a clear need to understand the transition from colonizer to invader, since the majority of S. aureus disease is the result of autoinfection from the colonized strain. Our recent findings indicate that the ArlRS TCS plays a critical role in the S. aureus transition to an invasive pathogen. The primary output of ArlRS is controlling the expression of MgrA, a cytoplasmic regulator that represses urease and large surface proteins and induces immune evasion factors. Our model is that S. aureus exhibits the traits of a commensal when MgrA levels are low, and exhibits the trait of an invasive pathogen when MgrA levels are high. To test this model, in Aim 1 we will determine the contribution of ArlRS to S. aureus adherence, colonization, and infection. Toward this end, we will compare WT, ∆arlRS, and ∆mgrA mutant strains using in vitro models of adherence and an in vivo model of skin colonization. Additionally, we will perform real-time in vivo imaging to track MgrA regulon expression, and carry out skin infection and immune evasion assessments of these strains. In Aim 2, we will perform a biochemical characterization of ArlS and identify of ArlR-dependent promoters. ArlR is a response regulator of the OmpR family, and the ArlR DNA- binding site and target promoters are unknown. The ArlS kinase has an N-terminal sensor domain with two membrane-spanning passes that flank a 14.1 kDa extracellular Cache domain. The goal of this aim is to determine the biochemical properties of ArlS and ArlR, which will provide critical insight into the mechanism of ArlS regulation and ArlR promoter targets. We will assess the kinetics of ArlS-ArlR phosphotransfer using mutants to determine the enzyme activity (kinase and phosphatase activity) that is important for regulating the ArlRS regulon. We will also use SELEX to identify the ArlR binding site and confirm target promoters with qRT- PCR and RNAseq. In Aim 3, we will perform molecular analysis of the ArlS sensing mechanism. We hypothesize that the ArlS extracellular Cache domain responds to an environmental cue that results in high urease and SasG expression to promote S. aureus colonization. To further investigate this mechanism, we will examine signal control over ArlS function using reporter strains, and we will make site-directed changes in ArlS Cache domain residues and assess function. We will also screen for additional ArlS Cache domain ligands from small-molecule libraries by thermal shift PCR assays and NMR. Discovering ligands that alter ArlRS function and prevent transition of S. aureus to an invasive pathogen could have potential in treating antibiotic-resistant infections.

项目摘要 金黄色葡萄球菌是一种条件致病菌，可引起广谱急性和慢性 感染.抗生素耐药性水平正在增长，耐甲氧西林的S。金黄色葡萄球菌（MRSA）感染是 治疗更具挑战性，导致患者和医疗保健系统的负担增加。尽管是 这种有效的病原体，S.金黄色葡萄球菌可以无症状地定殖约20%的健康成人 主要在鼻腔中，其次在皮肤上。显然需要了解 从殖民者到入侵者的转变，因为大多数S。金黄色葡萄球菌病是由于自身感染， 定殖菌株我们最近的研究结果表明，ArlRS TCS在S。金黄色转变 一种入侵性病原体ArlRS的主要输出是控制MgrA的表达， 抑制尿素酶和大的表面蛋白质并诱导免疫逃避因子的调节剂。我们的模式是 那辆多当MgrA水平低时，金黄色葡萄球菌表现出寄生虫的特征，并且表现出侵入性的特征。 当MgrA水平高时，病原体。为了测试该模型，在目标1中，我们将确定ArlRS对 S.金黄色葡萄球菌粘附、定殖和感染。为此，我们将比较WT、TumarlRS和TummgrA 使用粘附的体外模型和皮肤定殖的体内模型对突变菌株进行了研究。此外，我们将 进行实时体内成像，追踪MgrA调节子表达，进行皮肤感染和免疫， 对这些菌株的逃避评估。在目标2中，我们将对ArlS进行生化表征， ArIR依赖性启动子鉴定。ArlR是OmpR家族的反应调节剂，并且ArlR DNA- 结合位点和靶启动子是未知的。ArlS激酶具有N-末端传感器结构域，其具有两个 跨膜通道位于14.1 kDa细胞外Cache结构域的侧翼。这一目标的目的是 确定ArlS和ArlR的生物化学性质，这将为深入了解 ArlS调节和ArlR启动子靶点。我们将使用以下方法评估ArlS-ArlR磷酸转移的动力学： 突变体，以确定酶活性（激酶和磷酸酶活性），这是重要的调节 ArlRS调节子。我们还将使用SELEX来鉴定ArlR结合位点并用qRT-PCR确认靶启动子。 PCR和RNAseq.在目标3中，我们将进行ArlS传感机制的分子分析。我们假设 ArlS细胞外缓存结构域响应于导致高尿素酶和SasG的环境提示， 表达促进S.金黄色葡萄球菌定植。为了进一步研究这种机制，我们将研究信号 使用报告菌株控制ArlS功能，并且我们将在ArlS缓存结构域中进行定点改变 残留物和评估功能。我们还将从小分子中筛选额外的ArlS Cache结构域配体， 通过热移位PCR测定和NMR分析文库。发现改变ArlRS功能和预防ArlRS的配体 S的转变。金黄色葡萄球菌对侵入性病原体的耐受性可能在治疗耐药性感染方面具有潜力。